Genetic deficiency in factor VIII (fVIII), an essential component of the intrinsic pathway of blood coagulation, results in a life-threatening bleeding disorder Haemophilia A, which is treated by repeated infusions of expensive recombinant and plasma-derived fVIII products. The goal of the current application is to develop recombinant fVIII with a prolonged lifetime in circulation for more efficient therapy of Haemophilia A based on the breakthrough knowledge on the mechanisms of fVIII catabolism. We have established that fVIII clearance from circulation is mediated by low-density lipoprotein receptor-related protein (LRP), a member of LDL receptor superfamily, and facilitated by cell surface heparan sulfate proteoglycans (HSPGs). Simultaneous blocking of LRP and HSPGs in a mouse model, led to a significant, 5.5-fold prolongation of fVIII half-life. We have localized the major LRP- and HSPGs- binding sites within residues 484-509 and 558-565, respectively, of the A2 subunit of fVIII. These sites are exposed within fVIII complex with von Willebrand factor (vWf), in which fVIII is present in circulation. We propose to find an optimal combination of mutations within LRP- and HSPGs-binding sites and in their proximity, which would substantially reduce the corresponding components of fVIII clearance and will not affect the functional properties of fVIII. We will perform comprehensive site-specific mutagenesis using the isolated A2 subunit as a model of fVIII based on the identity of catabolism of A2 to that of fVIII from it complex with vWf. An optimal combination(s) of A2 mutations found to maximally reduce LRP- and HSPGs-mediated components of catabolism in a cell model without affecting the functional activity and stability of reconstituted activated fVIII, will be next introduced into fVIII constructs. We plan to use a construct encoding B domain-deleted fVIII, which provides high fVIII expression levels, and a fVIII construct carrying the B domain region 741-956, which is required for efficient fVIII secretion from the cell. We will apply a variety of methods to assess the ability of generated mutant fVIII to maintain interactions critical for its normal functioning, including ability for complex formation with vWf, interaction with components of the Xase complex, and normal activation/inactivation kinetics. We will also examine whether repeated use of mutant fVIII is not associated with increased immune response in fVIII-deficient mouse model of Haemophilia A. To obtain prognosis of the use of mutant fVIII in Haemophilia A patients, we will compare the ability of mutant and wild-type fVIII to stimulate in vitro proliferation of peripheral blood T lymphocytes from patients with severe Haemophilia A. Accomplishment of the current project will result in generation of mutant fVIII with prolonged lifetime in circulation, which will meet major functional, biochemical and immunological criteria.